Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations.G laucoma is the leading cause of irreversible blindness worldwide (1). It is a neurodegenerative disease in which vision loss is caused by the axonal injury and death of retinal ganglion cells (RGCs) (2), the projection neurons that process and transmit vision from the retina to the brain. Current therapies (i.e., surgery, laser, and eye drops) all act by lowering intraocular pressure (IOP). However, pressure reduction can be difficult to achieve, and even with significant pressure lowering, RGC loss can continue. Efforts have therefore been made to develop neuroprotective agents that would complement IOP-lowering therapies by directly inhibiting the RGC cell death process (3, 4). However, no neuroprotective agent has yet been approved for clinical use.Protein kinases provide attractive targets for the development of neuroprotective agents. A number of kinases, including cyclindependent kinases, death-associated protein kinases, JNK1-3, MAPKs, and glycogen synthase kinase-3β, are involved in neuronal cell death (5-12). An additional attraction is that protein kinases are readily druggable. The pharmacology and medicinal chemistry of kinase inhibitors are well-developed, with kinases now being the most important class of drug targets after G protein-coupled receptors (13). Although the primary clinical use of kinase inhibitors continues to be as antineoplastic agents, increasing attention is being paid to their use in other areas (14,15).To identify, in a comprehensive and unbiased manner, kinases that could serve as targets for neuroprotective glaucoma therapy, we screened the entire mouse kinome for kinases whose inhibition promotes RGC survival. For this screen, we develope...

Summary Dual leucine zipper kinase (DLK) has been implicated in cell death signaling secondary to axonal damage in retinal ganglion cells (RGCs) and other neurons. To better understand the pathway through which DLK acts, we developed enhanced functional genomic screens in primary RGCs, including use of arrayed, whole-genome, small interfering RNA libraries. Explaining why DLK inhibition is only partially protective, we identify leucine zipper kinase (LZK) as cooperating with DLK to activate downstream signaling and cell death in RGCs, including in a mouse model of optic nerve injury, and show that the same pathway is active in human stem cell-derived RGCs. Moreover, we identify four transcription factors (JUN), activating transcription factor 2 (ATF2), myocyte-specific enhancer factor 2A (MEF2A), and SRY-Box 11 (SOX11)) as being the major downstream mediators through which DLK/LZK activation leads to RGC cell death. Increased understanding of the DLK pathway has implications for understanding and treating neurodegenerative diseases.

This study demonstrates, for the first time, that SHIP2 is frequently downregulated in gastric cancer, and reduced SHIP2 expression promotes tumorigenesis and proliferation of gastric cancer via activation of the PI3K/Akt signaling.

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BackgroundThe aim of this study was to investigate the expression and silencing of the S100A8 gene, which encodes the S100 calcium-binding protein A8 (S100A8), and apoptosis and phosphorylation of protein kinase B (Akt) in tissue samples of endometrial carcinoma and HEC-1A endometrial adenocarcinoma cells in vitro.Material/MethodsImmunohistochemistry (IHC) was used to detect expression of the S100A8 protein in 74 tissue samples of endometrial cancer and 22 normal endometrial tissue samples. A stable S100A8 gene knockdown cell line was constructed using lentiviral packing short hairpin RNA (shRNA) transfected into HEC-1A cells. S100A8 mRNA and S100A8 protein levels were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting. The effects of expression of the S100A8 gene by endometrial cancer cells was investigated by the MTT assay, cell cycle and apoptotic assays, qRT-PCR, and Western blotting.ResultsIHC showed high levels of expression of S100A8 protein in endometrial carcinoma tissues, and HEC-1A adenocarcinoma cells (in G1 and G2). Increased expression of S100A8 protein was found endometrial cancer tissues compared with normal endometrial tissues (79.7% vs. 4.5%). S100A8 gene knockdown reduced cell proliferation in the HEC-1A cells compared with control cells, induced cell apoptosis, inhibited the phosphorylation of protein kinase B (Akt), and induced the expression of pro-apoptotic genes, including the cytochrome C gene, CYCS, BAD, BAX, FOXO1, FOXO3, CASP9, and CASP3.ConclusionsIn endometrial carcinoma cells, down-regulation of the S100A8 gene induced cell apoptosis via inhibition of the phosphorylated or active form of protein kinase B (Akt).

Background Generalized lymphatic anomaly is characterized by diffuse or multicentric proliferation of dilated lymphatic vessels resembling common lymphatic malformation. Studies on GLA are frequently hampered by a lack of appropriate models to test the effects of potential treatments or decipher the mechanism of pathology. Moreover, diverse phenotypes observed with GLA require a large number of samples to be analyzed to obtain statistically informative results. Due to the very limited experimental material, most of the research is restricted to single case report. Methods We first time used two-step endothelial cell isolation technique (step 1: single cells were first sorted with a-human CD31 magnetic beads; step 2: collected CD31 Pos cells from step1 were sorted with a-human PDPN magnetic beads) to generate two GLA-LEC cell lines, and purified normal-LEC from normal liver tissue in the same case. To characterize the aberrant phenotype of generalized lymphatic anomaly lymphatic endothelial cells (GLA-LEC#1, and GLA-LEC#2). We investigated GLA-LECs growth curve, cell cycle, apoptosis, and sprouting angiogenesis in vitro . Matrigel plug assay was applied in immunodeficient mice to monitor the GLA-LECs formed vasculature in vivo . Rapamycin and dual MEK / ERK inhibitor were tested to investigate the efficacy on inhibiting GLA-LEC proliferation and downstream signaling pathway. Results We have successfully purified GLA-LECs from GLA tissues with > 99% purity. These cells also expressed the lymphatic markers lymphatic vessel endothelial hyaluronan receptor (LYVE-1) and podoplanin (PDPN). GLA-LECs showed significantly higher proliferation rate compared to normal-LECs in both cases. Cell cycle analysis of cell distribution suggested that compared with normal-LECs, GLA-LECs showed increased proportion of cells in S phase and less G0/G1 phase. When GLA-LECs and normal-LECs apoptosis induced by serum deprivation, more Annexin V positive population of endothelial cells were observed in normal-LECs but not GLA-LECs. Hyper-activated epidermal growth-factor receptor (EGFR) signaling was observed in both cases of GLA-LECs, endogenously highly expression of EGF receptor and EGF induced phosphorylation of EGFR (phosphor Y1068) were found in both GLA cell lines. GLA-LECs are sensitive to both rapamycin and MEK / ERK dual inhibitor treatment. In vivo, by using Matrigel plug assay, we found both GLA-LECs and immortalized GLA-LEC (SV40) grew robust vessel-like structure. Conclusions In vitro , both GLA-LECs cell lines are highly proliferative as compared with normal-LECs. Rapamycin and dual MEK / ERK inhibitor dose-dependently inhibited GLA-LECs proliferation. In vivo , GLA-LECs showed angiogenic phenotype, and grew robust vessel-like structure in immunodeficient mice.